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The signaling lymphocytic activation molecule family 3 (SLAMF3) is highly expressed on plasma cells from patients with multiple myeloma (MM) and induces high malignant potential by ERK signaling mediated via the interaction with adaptor proteins SHP2 and GRB2. This study focused on the single-nucleotide polymorphism (SNP) of the SLAMF3 gene (rs509749, 1804A>G, M602V) in MM. The SNP G allele was a major type, and the frequencies of the GG, GA, and AA genotypes were 61.8%, 29.4%, and 8.8%, respectively, in patients with MM, which was almost the same as in healthy the control group in the Japanese population. Interestingly, patients with GG genotypes had significantly shorter overall survival times than patients with GA/AA genotypes. Consistent with those results, SLAMF3-overexpressing KMS-34 cells with the G allele (V602) had higher cell proliferation potential and were more resistant to anti-MM agents than those with the A allele (M602). When those cells were subcutaneously inoculated into NOG mice, tumor sizes in mice receiving V602 cells rapidly increased, and survival was significantly shorter than in mice injected with M602 cells. Furthermore, SLAMF3 V602 molecules bound more tightly to SHP2 and GRB2, with increased SHP2 and ERK phosphorylation compared with M602 cells. The mRNA expression of cell cycle-related genes (CCND1 and CCNE1) and anti-apoptotic genes (BCL2L and p21) was increased in V602 cells compared with M602 cells. The results thus suggested that the G allele of SLAMF3 SNP rs509749 may be associated with MM disease progression.

PD-L1 expressed on tumor cells contributes to disease progression with evasion from tumor immunity. Plasma cells from multiple myeloma (MM) patients expressed higher levels of PD-L1 compared with healthy volunteers and monoclonal gammopathy of undetermined significance (MGUS) patients, and its expression is significantly upregulated in relapsed/refractory patients. Furthermore, high PD-L1 expression is induced by the myeloma microenvironment and PD-L1+ patients with MGUS and asymptomatic MM tend to show disease progression. PD-L1 expression on myeloma cells was associated with more proliferative potential and resistance to antimyeloma agents because of activation of the Akt pathway through PD-1-bound PD-L1 in MM cells. Those data suggest that PD-L1 plays a crucial role in the disease progression of MM.

BCOR gene is a transcription regulatory factor that plays an essential role in normal hematopoiesis. The wider introduction of next-generation sequencing technology has led to reports in recent years of mutations in the BCOR gene in acute myeloid leukemia (AML), but the related clinical characteristics and prognosis are not sufficiently understood. We investigated the clinical characteristics and prognosis of 377 de novo AML cases with BCOR or BCORL1 mutation. BCOR or BCORL1 gene mutations were found in 28 cases (7.4%). Among cases aged 65 years or below that were also FLT3-ITD-negative and in the intermediate cytogenetic prognosis group, BCOR or BCORL1 gene mutations were observed in 11% of cases (12 of 111 cases), and this group had significantly lower 5-year overall survival (OS) (13.6% vs. 55.0%, P = 0.0021) and relapse-free survival (RFS) (14.3% vs. 44.5%, P = 0.0168) compared to cases without BCOR or BCORL1 gene mutations. Multivariate analysis demonstrated that BCOR mutations were an independent unfavorable prognostic factor (P = 0.0038, P = 0.0463) for both OS and RFS. In cases of AML that are FLT3-ITD-negative, aged 65 years or below, and in the intermediate cytogenetic prognosis group, which are considered to have relatively favorable prognosis, BCOR gene mutations appear to be an important prognostic factor.

We analyzed the clinical responses to thyrosine kinase inhibitors (TKIs) and the molecular and cytogenetic characteristics of 18 chronic myeloid leukemia (CML) patients with 3-way chromosomal translocations. The patients were 14 men and 4 women, aged 23-75 years (median 57 years). The Sokal risk was low in 12 patients, intermediate in 4 patients, and high in 2 patients. Newly identified translocation breakpoints were seen in 7 of the 18 patients. Three patients had the same breakpoints of t(9;22;11)(q34;q11.2;q23). The best responses to TKIs were partial cytogenic response (PCyR) in 2 patients, complete cytogenic response (CCyR) in 3 patients, molecular response (MR) 3.0 in 7 patients, MR 4.0 in 3 patients, and MR 4.5 or higher in 3 patients. A total of 66.7% of patients did not achieve MR 4.0 or higher. In 3 patients in whom TKIs resulted in MR 4.5 or higher for more than 2 years, TKI treatment was discontinued. However, all of them exhibited a loss of MR3.0, at 2, 6, and 20 months after the discontinuation of treatment, respectively, and TKI treatment needed to be restarted. According to Kaplan-Meier survival curve analysis, the overall survival (OS) was 100 months in 56% of the patients. The 60-months cumulative incidences of CCyR, MR3.0, MR4.0 and MR4.5 were 88.9%, 72.2%, 33.3%, and 16.7%, respectively. In the 11 analyzable patients, the BCR-ABL1 mRNA subtype was e14a2 type in 4 patients and e13a2 type in 7 patients.

Tyrosine kinase inhibitors (TKIs) improve the prognosis of patients with chronic myelogenous leukemia (CML) by inducing substantial deep molecular responses (DMRs); some patients could successfully discontinue TKI therapy after maintaining DMR for ≥1 year. In this cessation study, we investigated the optimal conditions for dasatinib discontinuation in patients who maintained DMR for ≥2 years. This study included 54 patients with CML who were enrolled in D-STOP multicenter prospective trial, had achieved DMR, and had discontinued dasatinib after 2-year consolidation. Peripheral lymphocyte profiles were analyzed by flow cytometry. The estimated 12-month treatment-free survival (TFS) was 62·9% (95% confidence interval: 48·5%–74·2%). During dasatinib consolidation, the percentage of total lymphocytes and numbers of CD3-CD56+ natural killer (NK) cells, CD16+CD56+ NK cells, and CD56+CD57+ NK-large granular lymphocytes (LGL) were significantly higher in patients with molecular relapse after discontinuation but remained unchanged in patients without molecular relapse for >7 months. At the end of consolidation, patients whose total lymphocytes comprised

TP53 gene abnormality has been reported to be an unfavorable prognostic factor in acute myeloid leukemia (AML). However, almost all studies of TP53 gene abnormality so far have been limited to mutation searches in the DNA binding domain. As there have been few reports examining both mutation and deletion over the full-length of the TP53 gene, the clinical characteristics of TP53 gene abnormality have not yet been clearly established. In this study, TP53 gene mutation was observed in 7.3% of the total 412 de novo AML cases (33 mutations in 30 cases), with mutation outside the DNA binding domain in eight cases (27%). TP53 gene deletion was observed in 3.1% of 358 cases. All cases had monoallelic deletion with TP53 gene mutation on the opposite allele. Multivariate analysis demonstrated that TP53 gene mutation in the DNA binding domain and outside the DNA binding domain was an independent poor prognostic factor for overall survival and relapse-free survival among the total cohort and it is also an unfavorable prognostic factor in FLT3-ITD-negative AML cases aged 70 years or below with intermediate cytogenetic prognosis. In stratified treatment, full-length search for TP53 gene mutation is therefore very important.

// Toshio Asayama 1 , Hideto Tamura 1 , Mariko Ishibashi 1 , Yasuko Kuribayashi-Hamada 1 , Asaka Onodera-Kondo 1 , Namiko Okuyama 1 , Akiko Yamada 1 , Masumi Shimizu 2 , Keiichi Moriya 1 , Hidemi Takahashi 2 and Koiti Inokuchi 1 1 Department of Hematology, Nippon Medical School, Tokyo, Japan 2 Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan Correspondence to: Hideto Tamura, e-mail: tam@nms.ac.jp Keywords: myelodysplastic syndromes, Tim-3, galectin-9, acute leukemia, immune checkpoint molecule Received: January 24, 2017 Accepted: August 23, 2017 Published: October 04, 2017 ABSTRACT T-cell immunoglobulin mucin-3 (Tim-3), an inhibitory immune checkpoint receptor, is highly expressed on acute myeloid leukemia cells and its ligand galectin-9 is reported to drive leukemic progression by binding with Tim-3. However, it remains unclear whether the Tim-3–galectin-9 pathway is associated with the pathophysiology of myelodysplastic syndromes (MDS). Thus, we investigated the expression and function of Tim-3 and the clinical impact of its ligand galectin-9 in MDS. Tim-3 expression levels on MDS blasts by CD45/side-scatter or CD34/CD45 gating were increased as MDS progressed to the advanced stage. Tim-3 expression in the MDS blasts was upregulated in the presence of the cell culture supernatant of human stromal cells or the MDS-related cytokine transforming growth factor-β1. The proliferation of Tim-3 + MDS blasts was inhibited by the blockade of anti-Tim-3 antibody. Furthermore, plasma levels of galectin-9 were elevated as MDS progressed to the advanced stage in 70 MDS/acute leukemia transformed from MDS patients and was a prognostic factor in 40 MDS patients. Our data demonstrated that the Tim-3-galectin-9 pathway is associated with the pathogenesis and disease progression of MDS. These findings provide new insight into potential immunotherapy targeting the galectin-9–Tim-3 pathway in MDS.

Acute lymphoblastic leukemias (ALL) positive for KMT2A/AFF1 (MLL/AF4) translocation, which constitute 60% of all infant ALL cases, have a poor prognosis even after allogeneic hematopoietic stem cell transplantation (allo-HSCT). This poor prognosis is due to one of two factors, either resistance to TNFα, which mediates a graft-versus-leukemia (GVL) response after allo-HSCT, or immune resistance due to upregulated expression of the immune escape factor S100A6. Here, we report an immune stimulatory effect against KMT2A/AFF1-positive ALL cells by treatment with the anti-allergy drug amlexanox, which we found to inhibit S100A6 expression in the presence of TNF-α. In KMT2A/AFF1-positive transgenic (Tg) mice, amlexanox enhanced tumor immunity and lowered the penetrance of leukemia development. Similarly, in a NOD/SCID mouse model of human KMT2A/AFF1-positive ALL, amlexanox broadened GVL responses and extended survival. Our findings show how amlexanox degrades the resistance of KMT2A/AFF1-positive ALL to TNFα by downregulating S100A6 expression, with immediate potential implications for improving clinical management of KMT2A/AFF1-positive ALL. Cancer Res; 77(16); 4426–33. ©2017 AACR.

We previously showed that the interaction of programmed death-ligand 1 (PD-L1) on multiple myeloma (MM) cells with PD-1 not only inhibits tumor-specific cytotoxic T-lymphocyte activity via the PD-1 signaling pathway but also induces drug resistance via PD-L1–mediated reverse signals. We here examined the regulation of PD-L1 expression by immunomodulatory drugs (IMiDs) and antimyeloma effects of the anti–PD-L1 antibody durvalumab in combination with IMiDs. IMiDs induced PD-L1 expression on IMiD-insensitive MM cells and plasma cells from patients newly diagnosed with MM. Gene-expression profiling analysis demonstrated that not only PD-L1, but also a proliferation-inducing ligand (APRIL), was enhanced by IMiDs. PD-L1 induction by IMiDs was suppressed by using the APRIL inhibitor recombinant B-cell maturation antigen (BCMA)-Ig, the antibody against BCMA, or an MEK/ERK inhibitor in in vitro and in vivo assays. In addition, its induction was abrogated in cereblon (CRBN)-knockdown MM cells, whereas PD-L1 expression was increased and strongly induced by IMiDs in Ikaros-knockdown cells. These results demonstrated that PD-L1 upregulation by IMiDs on IMiD-insensitive MM cells was induced by (i) the BCMA–APRIL pathway via IMiD-mediated induction of APRIL and (ii) Ikaros degradation mediated by CRBN, which plays a role in inhibiting PD-L1 expression. Furthermore, T-cell inhibition induced by PD-L1–upregulated cells was effectively recovered after combination treatment with durvalumab and IMiDs. PD-L1 upregulation by IMiDs on MM cells might promote aggressive myeloma behaviors and immune escape in the bone marrow microenvironment.

This study presents the final report of the multicenter, prospective tyrosine kinase inhibitor discontinuation study, D‐STOP, after a 3‐year follow‐up of 54 patients with chronic CML who discontinued dasatinib after a sustained deep molecular response (DMR) for ≥2 years with dasatinib treatment. Estimated treatment‐free remission (TFR) rates at 12 and 36 months were 63.0% [95% confidence interval (CI): 48.7‐74.3] and 59.3% (95% CI: 45.0‐71.0), respectively. CD3−CD56+ NK, CD16+CD56+ NK, and CD57+CD56+ NK large granular lymphocyte (NK‐LGL), CD8+CD4– cytotoxic T cell, and CD57+CD3+ T‐LGL cell numbers were relatively elevated throughout the 24‐month consolidation only in failed patients who molecularly relapsed within 12 months. In successful patients, these subsets elevated transiently after 12 months, but returned to basal levels after 24‐month consolidation. Therefore, smaller changes in NK/T, particularly the NK subset throughout consolidation, reflected higher TFR rates. TFR rates of those patients exhibiting elevation in CD3−CD56+ NK >376 cells/μL, CD16+CD56+ NK > 241 cells/μL, or CD57+CD56+ NK‐LGL >242 cells/μL during consolidation compared with others were 26.7% (8.3%‐49.6%) vs 78.3% (55.4%‐90.3%), HR 0.032 (0.0027‐0.38; P = .0064), 31.2% (11.4%‐53.6%) vs 85.0% (60.4%‐94.9%), HR 0.039 (0.0031‐0.48; P = .011), or 36.8% (16.5%‐57.5%) vs 77.3% (53.7%‐89.8%), HR 0.21 (0.065‐0.69; P = .010), respectively. Therefore, silent responses of T/NK subsets to dasatinib throughout consolidation were significant for longer TFR. Elevated NK/T, particularly NK lymphocytes responsive to dasatinib, may be immunologically insufficient to maintain TFR. Their decline, subsequently replaced by altered lymphocyte population with less response to dasatinib during sustained DMR, might be immunologically significant. (D‐STOP, NCT01627132)., The figure shows that silent NK/T cell reactions against dasatinib, particularly that of NK cells, during sustained DMR before dasatinib discontinuation are significant for longer treatment‐free remission.

Despite therapeutic advances over the past decades, multiple myeloma (MM) remains a largely incurable disease with poor prognosis in high-risk patients, and thus new treatment strategies are needed to achieve treatment breakthroughs. MM represents various forms of impaired immune surveillance characterized by not only disrupted antibody production but also immune dysfunction of T, natural killer cells, and dendritic cells, although immunotherapeutic interventions such as allogeneic stem-cell transplantation and dendritic cell-based tumor vaccines were reported to prolong survival in limited populations of MM patients. Recently, epoch-making immunotherapies, i.e., immunomodulatory drug-intensified monoclonal antibodies, such as daratumumab combined with lenalidomide and chimeric antigen receptor T-cell therapy targeting B-cell maturation antigen, have been developed, and was shown to improve prognosis even in advanced-stage MM patients. Clinical trials using other antibody-based treatments, such as antibody drug-conjugate and bispecific antigen-directed CD3 T-cell engager targeting, are ongoing. The manipulation of anergic T-cells by checkpoint inhibitors, including an anti-T-cell immunoglobulin and ITIM domains (TIGIT) antibody, also has the potential to prolong survival times. Those new treatments or their combination will improve prognosis and possibly point toward a cure for MM.

The signaling lymphocytic activation molecule family 3 (SLAMF3) is a member of the immunoglobulin superfamily expressed on T, B, and natural killer cells and modulates the activation and cytotoxicity of these cells. SLAMF3 is also expressed on plasma cells from patients with multiple myeloma (MM), although its role in MM pathogenesis remains unclear. This study found that SLAMF3 is highly and constitutively expressed on MM cells regardless of disease stage and that SLAMF3 knockdown/knockout suppresses proliferative potential and increases drug-induced apoptosis with decreased levels of phosphorylated ERK protein in MM cells. SLAMF3-overexpressing MM cells promote aggressive myeloma behavior in comparison with cytoplasmic domain-truncated SLAMF3 (ΔSLAMF3) cells. SLAMF3 interacts directly with adaptor proteins SH2 domain-containing phosphatase 2 (SHP2) and growth factor receptor bound 2 (GRB2), which also interact with each other. SLAMF3 knockdown, knockout, ΔSLAMF3, and SHP2 inhibitor-treated MM cells decreased phosphorylated ERK protein levels. Finally, serum soluble SLAMF3 (sSLAMF3) levels were markedly increased in advanced MM. Patients with high levels of sSLAMF3 progressed to the advanced stage significantly more often and had shorter progression-free survival times than those with low levels. This study revealed that SLAMF3 molecules consistently expressed on MM cells transmit MAPK/ERK signals mediated via the complex of SHP2 and GRB2 by self-ligand interaction between MM cells and induce a high malignant potential in MM. Furthermore, high levels of serum sSLAMF3 may reflect MM disease progression and be a useful prognostic factor. Implications: SLAMF3 may be a new therapeutic target for immunotherapy and novel agents such as small-molecule inhibitors.

B7 homolog 1 (B7-H1)–expressing myeloma cells not only inhibit myeloma-specific cytotoxic T lymphocytes (CTL), but also confer a proliferative advantage: resistance to antimyeloma chemotherapy. However, it remains unknown whether B7-H1 expressed on myeloma cells induces cellular responses associated with aggressive myeloma behaviors. To address this question, we analyzed the proliferation and drug sensitivity of B7-H1–expressing myeloma cells transfected with B7-H1–specific short-hairpin RNA or treated with programmed cell death (PD)-1-Fc–coupled beads. Knockdown of B7-H1 expression in myeloma cells significantly inhibited cell proliferation and increased apoptosis induced by the chemotherapeutic alkylating agent melphalan, with downregulation of the expression of cell cycle–related genes (CCND3 and CDK6) and antiapoptotic genes (BCL2 and MCL1). B7-H1 molecules thus contributed to myeloma cell-cycle progression and suppression of drug-induced apoptosis. B7-H1–expressing myeloma cells had a higher affinity for PD-1 than for CD80. PD-1-Fc bead–treated myeloma cells also became resistant to apoptosis that was induced by melphalan and the proteasome inhibitor bortezomib. Apoptosis resistance was associated with the PI3K/AKT pathway. Both myeloma cell drug resistance and antiapoptotic responses occurred through the PI3K/AKT signaling pathway, initiated from “reverse” stimulation of B7-H1 by PD-1. Therefore, B7-H1 itself may function as an oncogenic protein in myeloma cells. The interaction between B7-H1 on myeloma cells and PD-1 molecules not only inhibits tumor-specific CTLs but also induces drug resistance in myeloma cells through the PI3K/AKT signaling pathway. These observations provide mechanistic insights into potential immunotherapeutic benefits of blocking the B7-H1–PD-1 pathway. Cancer Immunol Res; 4(9); 779–88. ©2016 AACR.

The risk of complication of polycythemia vera (PV) and essential thrombocythemia (ET) by thrombosis in Japanese patients is clearly lower than in western populations, suggesting that genetic background such as race may influence the clinical features. This study aimed to clarify the relationship between genetic mutations and haplotypes and clinical features in Japanese patients with PV and ET. Clinical features were assessed prospectively among 74 PV and 303 ET patients. There were no clinical differences, including JAK2V617F allele burden, between PV patients harboring the various genetic mutations. However, CALR mutation-positive ET patients had a significantly lower WBC count, Hb value, Ht value, and neutrophil alkaline phosphatase score (NAP), and significantly more platelets, relative to JAK2V617F-positive ET patients and ET patients with no mutations. Compared to normal controls, the frequency of the JAK246/1 haplotype was significantly higher among patients with JAK2V617F, JAK2Ex12del, or MPL mutations, whereas no significant difference was found among CALR mutation-positive patients. CALR mutation-positive patients had a lower incidence of thrombosis relative to JAK2V617F-positive patients. Our findings suggest that JAK2V617F-positive ET patients and CALR mutation-positive patients have different mechanisms of occurrence and clinical features of ET, suggesting the potential need for therapy stratification in the future.

Bendamustine is a novel alkylator containing a benzimidazole ring, which adds purine analogue-like properties in addition to the alkylating properties.[1] Several clinical studies have showed that ...

Acute myeloid leukemia (AML) with an FLT3 internal tandem duplication (FLT3-ITD) mutation is an aggressive hematologic malignancy with a grave prognosis. To identify the mutational spectrum associated with relapse, whole-exome sequencing was performed on 13 matched diagnosis, relapse, and remission trios followed by targeted sequencing of 299 genes in 67 FLT3-ITD patients. The FLT3-ITD genome has an average of 13 mutations per sample, similar to other AML subtypes, which is a low mutation rate compared with that in solid tumors. Recurrent mutations occur in genes related to DNA methylation, chromatin, histone methylation, myeloid transcription factors, signaling, adhesion, cohesin complex, and the spliceosome. Their pattern of mutual exclusivity and cooperation among mutated genes suggests that these genes have a strong biological relationship. In addition, we identified mutations in previously unappreciated genes such as MLL3, NSD1, FAT1, FAT4, and IDH3B. Mutations in 9 genes were observed in the relapse-specific phase. DNMT3A mutations are the most stable mutations, and this DNMT3A-transformed clone can be present even in morphologic complete remissions. Of note, all AML matched trio samples shared at least 1 genomic alteration at diagnosis and relapse, suggesting common ancestral clones. Two types of clonal evolution occur at relapse: either the founder clone recurs or a subclone of the founder clone escapes from induction chemotherapy and expands at relapse by acquiring new mutations. Relapse-specific mutations displayed an increase in transversions. Functional assays demonstrated that both MLL3 and FAT1 exert tumor-suppressor activity in the FLT3-ITD subtype. An inhibitor of XPO1 synergized with standard AML induction chemotherapy to inhibit FLT3-ITD growth. This study clearly shows that FLT3-ITD AML requires additional driver genetic alterations in addition to FLT3-ITD alone.

We conducted a comprehensive analysis of 28 recurrently mutated genes in acute myeloid leukemia (AML) in 271 patients with de novo AML. Co-mutations were frequently detected in the intermediate cytogenetic risk group, at an average of 2.76 co-mutations per patient. When assessing the prognostic impact of these co-mutations in the intermediate cytogenetic risk group, overall survival (OS) was found to be significantly shorter (P=0.0006) and cumulative incidence of relapse (CIR) significantly higher (P=0.0052) in patients with complex molecular genetic abnormalities (CMGAs) involving three or more mutations. This trend was marked even among patients aged ⩽65 years who were also FLT3-ITD (FMS-like tyrosine kinase 3 internal tandem duplications)-negative (OS: P=0.0010; CIR: P=0.1800). Moreover, the multivariate analysis revealed that CMGA positivity was an independent prognostic factor associated with OS (P=0.0007). In stratification based on FLT3-ITD and CEBPA status and 'simplified analysis of co-mutations' using seven genes that featured frequently in CMGAs, CMGA positivity retained its prognostic value in transplantation-aged patients of the intermediate cytogenetic risk group (OS: P=0.0002. CIR: P

We evaluated the effects of regulatory T cell (Treg) inhibition during dasatinib treatment on the anticancer immune response, particularly on natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Fifty-two newly diagnosed Japanese patients with chronic myeloid leukemia (CML) in the chronic phase were enrolled in the D-first study; all received 100 mg of dasatinib once daily and were followed for at least 36 months. The cumulative deep molecular response (DMR, MR4) rate was 65% by 36 months; the 3-year overall survival was 96%. CD4+ T cell counts were stable, whereas the proportion of CD4+CD25+CD127low (Treg) cells decreased in a time-dependent manner. The DMR rate by18 months was significantly better in low Treg patients (

High PRDM16 (also known as MEL1) expression is a representative marker of acute myeloid leukemia (AML) with NUP98-NSD1 and is a significant predictive marker for poor prognosis in pediatric AML. However, the clinical features of adult AML with PRDM16 expression remain unclear. PRDM16 is highly homologous to MDS1/EVI1, which is an alternatively spliced transcript of MECOM (also known as EVI1). We investigated PRDM16 expression in 151 AML patients, with 47 (31%) exhibiting high PRDM16 expression (PRDM16/ABL1 ratio ≥ 0.010). High PRDM16 expression significantly correlated with DNMT3A (43% vs. 15%, P

Background: As recurrence of acute myeloid leukemia (AML) is difficult to predict, it is important to detect it by measuring minimal residual disease (MRD). PML-RARA, RUNX-RUNX1T1, CBFB-MYH11 are regarded as the reliable MRD markers. However, in AML with normal karyotype and many other forms, no MRD markers have been established. NPM1 mutations, occurring in approximately 30% of adult AML cases, and 50-60% of AML cases with normal karyotype, represent one of the most frequent mutations in AML. Recently, NPM1 mutation is reported to be useful in assessing MRD. We undertook a retrospective and prospective investigation of the usefulness of NPM1 mutation as an MRD marker in Japanese patients with AML. Methods: The subjects were 38 NPM1-mutated AML patients with first hematological remission at several hospitals related to our institution between 2001 and 2018. This study was approved by the ethics committee of Nippon Medical School and the informed consents were obtained from all patients, according to the Declaration of Helsinki. We analyzed peripheral blood cells or bone marrow cells at diagnoses, and evaluated only bone marrow cells after diagnoses. Detection of NPM1 mutation was carried out using allele-specific real time PCR following creation of a complementary primer. After dilution of the samples, sensitivity to TCTG, CATG, and CCTG was found to be 0.001%. The NPM1 mutant copies were qualified only at successful amplification of internal control. Results: The median age of the patients was 58 years (18-79 years). There were 32 cases with intermediate cytogenetic prognosis and 6 cases with unclear chromosomal profile. Of the 38 cases, 14 cases (37%) were FLT3-ITD-positive and allogeneic hematopoietic stem cell transplantation was carried out in 14 cases (37%). The base sequence was TCTG in 36 cases and CCTG in 2 cases. Persistence of NPM1-mutatation was present in 25 patients with first hematological remission (66%). Compared with patients with MRD negative, patients with MRD positive were associated with DNMT3A mutation (MRD positive 12/25 vs MRD negative 0/13, p=0.003). The rate of relapse in patients with MRD positive was significantly higher than those of in patients with MRD negative (MRD positive 76% vs MRD negative 23%, p=0.004). The rates of relapse free survival (RFS) and overall survival (OS) in patients with MRD positive were significantly lower than those in patients with MRD negative (RFS at 2 years: MRD positive 14% vs MRD negative 86% p=0.003; Figure 1, OS at 2 years: MRD positive 25% vs MRD negative 93%, p Conclusion: The presence of MRD with NPM1 mutation is significantly associated with relapse and it is useful to decide their treatment strategy. Especially, there is the usefulness of NPM1 mutation as an MRD marker in NPM1 positive Flt3-ITD negative AML patients who are generally classified as favorable risk. According to previous reports, it is known that NPM1-mutated AML sometimes relapse with losing NPM1 mutations. However, in this study, all NPM1-mutated AML relapse without losing NPM1 mutations. We need to collect more patients and are going to confirm whether there are patients who relapse with losing NPM1 mutations or not. We plan to analyze the genetic background of MRD positive and negative patients with next-generation sequencing. We are going to announce the genetic characteristics in addition to this result at ASH. Disclosures Usuki: Astellas Pharma Inc: Research Funding, Speakers Bureau; Daiichi Sankyo Co., Ltd.: Research Funding, Speakers Bureau. Kako:Bristol-Myers Squibb: Honoraria; Pfizer Japan Inc.: Honoraria. Inokuchi:Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria; Celgene: Honoraria; Pfizer: Honoraria.

Introduction: We previously reported that CD86 expressed on tumor cells from multiple myeloma (MM) patients is associated with a proliferative advantage of tumor cells and suppresses the antitumor immune response by inducing the immunosuppressive cytokine IL-10 from CD4+ T cells via CD86-CD28 interaction [Yamashita T, Clin Cancer Res 2009]. Furthermore, CD28 expressed on MM cells can mediate pro-survival signaling through CD86-CD28 interaction, resulting in chemotherapeutic resistance [Murray ME, Blood 2014]. Hock et al. reported that serum soluble CD86 levels (sCD86) were a significant independent prognostic marker in MM patients treated with conventional chemotherapy [Br J Haematol 2006]. However, it is unknown whether serum soluble CD86 is still a prognostic marker in the novel agent era and how sCD86 is produced in serum. In this study, we investigated the association of clinical characteristics and prognosis with serum sCD86 and elucidated the mechanism by which sCD86 is produced in MM patients. Materials and Methods: 1) Peripheral blood and bone marrow (BM) samples were obtained from 294 newly diagnosed (42 asymptomatic and 252 symptomatic) MM patients, 16 patients with monoclonal gammopathy of undetermined significance (MGUS), and 16 healthy controls. 2) sCD86 levels were measured using ELISA. 3) The expression of cell-surface antigen on plasma cells identified as CD138-positive and CD38-strong positive cells were analyzed using flow cytometry. Expression of the CD86 full-length (variant 1) and alternatively spliced variant deleting exon 6 (variant 3), which encodes for the transmembrane domain, in MM cell lines and CD138+ plasma cells isolated from BM samples from MM patients were analyzed using real-time PCR. Results: 1) sCD86 levels were significantly increased in MM patients compared with MGUS patients and healthy controls. Among MM patients, the levels were significantly higher in symptomatic than in asymptomatic patients and markedly increased in advanced stages (International Staging System [ISS] stage II/III and revised ISS II/III). We next investigated the differences in clinical characteristics between two groups according to serum sCD86 levels: high (≥2.16 ng/mL); and low ( Conclusions: We demonstrated that sCD86 is produced via expression of CD86 variant 3 in primary MM cells and that sCD86 is a prognostic marker in the novel agent era. These results suggest that serum sCD86 levels may reflect disease progression through CD28 expressed on myeloma cells and be a useful prognostic factor. Disclosures Inokuchi: Novartis: Honoraria; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria, Research Funding; Pfizer: Honoraria. Handa:Ono: Research Funding. Komatsu:Fuso Pharmaceutical Industries, Ltd.: Research Funding; Novartis K.K: Speakers Bureau; Pharma Essentia: Research Funding, Speakers Bureau; Wako Pure Chemical Industries, Ltd.: Research Funding; Takeda Pharmaceutical Company Limited: Research Funding, Speakers Bureau. Imai:Celgene: Honoraria, Research Funding; Janssen Parmaceutical K.K.: Honoraria, Research Funding; Bristol-Myers Squibb: Research Funding. Ito:Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ono: Honoraria. Tamura:Celegene: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria; Ono Pharmaceutical: Honoraria; Takeda Pharmaceutical: Honoraria.

Background With the introduction of imatinib, a first-generation tyrosine kinase inhibitor (TKI) to inhibit BCR-ABL1 kinase, the outcome of chronic-phase chronic myeloid leukemia (CP-CML) has improved dramatically. However, only a small proportion of CP-CML patients subsequently achieve a deep molecular response (DMR) with imatinib. Dasatinib, a second-generation TKI, is more potent than imatinib in the inhibition of BCR-ABL1 tyrosine kinase in vitro and more effective in CP-CML patients who do not achieve an optimal response with imatinib treatment. Methods In the present study, we attempted to investigate whether switching the treatment from imatinib to dasatinib can induce DMR in 16 CP-CML patients treated with imatinib for at least two years who achieved a major molecular response (MMR) with detectable levels of BCR-ABL1 transcripts. Results The rates of achievement of DMR at 1, 3, 6 and 12 months after switching to dasatinib treatment in the 16 patients were 44% (7/16), 56% (9/16), 63% (10/16) and 75% (12/16), respectively. The cumulative rate of achieving DMR at 12 months from initiation of dasatinib therapy was 93.8% (15/16). The proportion of natural killer cells and cytotoxic T cells in peripheral lymphocytes increased after switching to dasatinib. In contrast, the proportion of regulatory T cells decreased during treatment. The safety profile of dasatinib was consistent with previous studies. Conclusion Switching to dasatinib would be a therapeutic option for CP-CML patients who achieved MMR but not DMR by imatinib, especially for patients who wish to discontinue TKI therapy.

Gene mutations were found in acute myeloid leukemia (AML) and their importance has been noted. To clarify the importance and stability of mutations, we examined gene mutations in paired samples at diagnosis and relapse of 34 adult AML patients. Five acquired gene mutations were detected at relapse. Of the 45 gene mutations at diagnosis, 11 of them were lost at relapse. The acquired mutations at relapse were all class I mutations as Fms-like tyrosine kinase 3 (FLT3) and rat sarcoma viral oncogene homolog (RAS) mutations. The disappeared mutations at relapse were 3 of 11 internal tandem duplications of FLT3 (FLT3-ITD) (27.3%), 3 of 3 FLT3 tyrosine kinase domain (FLT3-TKD) (100%), 3 of 13 Nucleophosmin 1 (23.1%) and 2 of 5 CCAAT/enhancer-binding protein-α (40%) mutations. However, epigenetics-modifying gene (DNMT3a, TET2 and IDH1/2) mutations had no change between diagnosis and relapse samples, and may become minimal residual disease marker. The frequency of FLT3-ITD at relapse in patients with DNMT3a mutation at diagnosis is significantly higher than those in patients without them (P=0.001). Moreover, the high frequency of FLT3-ITD at relapse is also seen in AML cases that initially present with any epigenetics-modifying gene mutations (P

The TARGET system is an online database that can be easily accessed by physicians. The registration of one's own chronic myeloid leukemia (CML) patients in the TARGET system makes it possible to share experiences among physicians, and, thus, may facilitate appropriate treatment for patients. Patients were registered in the TARGET system from October 2003 to March 2010 in Japan. A total of 1236 patients from 176 hospitals were registered in Japan. We analyzed data from 639 CML chronic phase patients not receiving prior therapy registered in this system. After 90 months follow-up, high survival rates were demonstrated for imatinib-treated newly diagnosed CML patients, with event-free survival (EFS), progression-free survival (PFS), and overall survival (OS) rates of 79.1, 94.8, and 95.1%, respectively. A landmark analysis of 296 patients who showed a complete cytogenetic response (CCyR) at 12 months after the initiation of imatinib treatment revealed that, at 90 months, 99% of patients (95% CI, 98-100) had not progressed to accelerated phase (AP) or blastic crisis (BC). The patients showing a CCyR and a reduction of at least 3log levels of BCR-ABL transcripts after 18 months of treatment had an estimated survival rate without CML progression of 100% at 84 months. The probability of achieving undetectable BCR-ABL in patients by 72 months with an major molecular response (MMR) at 12 months was 86.5%, compared with 64.7% for those without an MMR (p < 0.0001). There were no new safety issues. In summary, based on this 7-year TARGET analysis, imatinib showed a continual clinical benefit as first-line therapy for newly diagnosed CML. The TARGET system may represent a more practical and general feature compared with the IRIS study.

Rearrangements of the MLL gene located at 11q23 are common chromosomal abnormalities associated with acute leukemia (AL), especially infant and secondary leukemia after previous treatment with DNA topoisomerase II inhibitors. 11q23/MLL abnormalities have been widely recognized as an important prognostic factor in AL. Over 70 chromosome partners of 11q23 have been identified to date, at least 50 of which have been cloned and characterized at the molecular level. Recent studies showed that the prognosis of 11q23/MLL AL varies widely according to the partner gene, the leukemia cell lineage, the age of the patient and the treatment administered. Special strategies are needed to treat 11q23/MLL AL, including allogeneic hematopoietic stem cell transplantation, according to the fusion partner. The development of novel methodologies, including new molecular therapeutic targets, is also needed to improve the prognosis of 11q23/MLL AL. The present article provides an update on the current status of prognosis and treatment of 11q23/MLL AL according to the fusion partner.

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

Objective Concerning MLL-AF4 leukemogenesis, previous mouse models suggest that the tumorigenesis capacity of MLL-AF4 alone is insufficient for causing leukemia. Based on the finding that an Fms-like tyrosine kinase 3 (Flt3) gene mutation in the tyrosine kinase domain (TKD) was observed in approximately 15% of mixed lineage leukemia (MLL), we investigated synergistic leukemogenesis effects of the two genes in vitro. Materials and Methods In a mouse interleukin-3 (IL-3)−dependent cell line, 32Dc, expression of MLL-AF4 and mutant Flt3 was induced using a lentiviral vector. We analyzed apoptosis induction in the absence of IL-3 and the granulocyte colony-stimulating factor−related induction of differentiation, gene expression profiling, and the mechanism involved in the synergistic effects of MLL-AF4 and Flt3-TKD. Results Neither Flt3-expressing 32Dc (32Dc Flt3-TKD ) nor MLL-AF4−expressing 32Dc (32Dc MLL-AF4 ) acquired IL-3−independent proliferative capacity in semisolid/liquid media. However, Flt3-TKD+MLL-AF4−expressing 32Dc (32Dc Flt3-TKD+MLL-AF4 ) acquired a non−IL-3−dependent proliferative capacity by inhibiting apoptosis in the two media. The 32Dc Flt3-TKD and 32Dc MLL-AF4 cells differentiated into granulocytes in the presence of granulocyte colony-stimulating factor. However, in the 32Dc Flt3-TKD+MLL-AF4 cells, there was no differentiation. Subsequently, we performed gene expression profiling. The enhancement of Hox genes expression was not identified. However, expression of S100A6 was synergistically enhanced in the presence of both MLL-AF4 and Flt3-TKD genes. Moreover, anti-S100A6 small interfering RNA downregulated leukemic proliferation. Conclusion We conclude that their synergistic enhancement of S100A6 expression plays an important role in MLL-AF4−associated leukemogenesis.

In recent years, it has been reported that DNA-methylation regulatory gene mutation - mutation of the genes that regulate gene expression through DNA methylation - is observed with high frequency in acute myeloid leukemia. The objective of the present study was to elucidate the clinical characteristics and prognosis of acute myeloid leukemia with associated DNA-methylation regulatory gene mutation. The subjects were 308 patients with acute myeloid leukemia. DNA-methylation regulatory gene mutation was observed in 135 of the 308 cases (43.8%). acute myeloid leukemia associated with DNA-methylation regulatory gene mutation was more frequent in older patients ( p

Antiangiogenic gene therapy offers an attractive approach to the treatment of a variety of malignancies, including those of the hematological system. However, evaluation of this approach has been hampered by the lack of appropriate animal models. We have recently produced transgenic mice expressing P230 bcr/abl that develop myeloproliferative disease (MPD) closely resembling human chronic myelogenous leukemia. Using this MPD murine model, we examined the feasibility of systemic antiangiogenic gene therapy for hematological malignancy. An adenoviral vector containing the secretable endostatin gene was injected into the right quadriceps muscle of the MPD mice. The increased endostatin level was detected for at least 6 months. Hematological parameters including platelet counts, granulocyte counts, and the hemoglobin concentration were improved by this gene therapy. Infiltration of megakaryocytes was also significantly inhibited in treated MPD mice. Reduction of the microvessel density was confirmed by histological examination. These results demonstrated, for the first time, that antiangiogenic gene therapy is effective to inhibit leukemogenesis caused by expression of the chimeric bcr/abl gene.

We reported here abnormal expression profile of the bcl-x gene is associated with the recurrence in acute myelogenous leukemia (AML).The bcl-x gene product exists in two forms, bcl-xl and bcl-xs. The bcl-xl, similar to bcl-2, inhibits apoptosis, whereas bcl-xs counters the effects of bcl-2 and bcl-xl. Mononuclear cells of bone marrow cells were obtained from 50 patients with recurrent AML at diagnosis prior to treatment and during relapse. We investigated the expression of the two isoforms of the bcl-x gene, bcl-xl and bcl-xs. The bcl-xl transcript was detected in all patients at both stages, but the bcl-xs transcript was not detected in six cases at both stages and in seven other cases at relapse (bcl-xs (-) group, n=13). The duration of relapse-free survival (RFS) and overall survival (OS) of the bcl-xs (-) group were significantly shorter than those of the bcl-xs (+) group (RFS: P=0.04; OS: P=0.02). Subsequently, mutation of the bcl-x gene was investigated. No mutation of the bcl-x gene was detected in all patients of both stages. Some refractory leukemic cells which escape chemotherapy-induced apoptosis due to loss of the bcl-xs transcript may continue to proliferate, resulting in the relapse of chemotherapy-resistant leukemia. The examination of the expression status of the bcl-x gene could facilitate the prediction of chemotherapy resistance and the prognosis of patients with refractory AML. The future large study will confirm the present data about loss of bcl-xs gene expression as prognostic factor.

Chronic myelogenous leukemia (CML) is characterized by the Philadelphia (Ph) chromosome and bcr/abl gene rearrangement which occurs in pluripotent hematopoietic progenitor cells expressing the c-kit receptor tyrosine kinase (KIT). To elucidate the biological properties of KIT in CML leukemogenesis, we performed analysis of alterations of the c-kit gene and functional analysis of altered KIT proteins. Gene alterations in the c-kit juxtamembrane domain of 80 CML cases were analyzed by reverse transcriptase and polymerase chain reaction-single strand conformation polymorphism (RT-PCR-SSCP). One case had an abnormality at codon 564 (AAT --AAG, Asn --Lys), and six cases had the same base abnormality at codon 541 (ATG --CTG, Met --Leu) in the juxtamembrane domain. Because the change from Met to Leu at codon 541 was a conservative one which was also observed in the normal population and normal tissues of CML patients, it probably represents a polymorphic variation. Although samples of hair roots and leukemic cells from the chronic phase of one CML patient showed no abnormality, an abnormality at codon 541 (ATG --CTG, Met --Leu) was found only at blastic crisis (BC) of this case. In the case with the abnormality at codon 564, the mutation was detected only in a sample of leukemic cells collected at BC. To examine the biological consequence and biological significance of these abnormalities, murine KIT(L540) and KIT(K563) expression vectors were introduced into interleukin-3 (IL-3)-dependent murine Ba/F3 cells to study their state of tyrosine phosphorylation and their growth rate. Ba/F3 cells expressing KIT(WT), KIT(L540) and KIT(K563) showed dose-dependent tyrosine phosphorylation after treatment with increasing concentrations of recombinant mouse stem cell factor (rmSCF). The cells expressing KIT(L540) and KIT(K563) were found to have greater tyrosine phosphorylation than cells expressing KIT(WT) at 0.1 and 1.0 ng/ml of rmSCF. The Ba/F3 cells expressing KIT(K563) proliferated in response to 0.1 and 1.0 ng/ml of rmSCF as well as IL-3. The Ba/F3 cells expressing KIT(L540)showed a relatively higher proliferative response to 0.1 ng/ml of rmSCF than the response of cells expressing KIT(WT). These mutations and in vitro functional analyses raise the possibility that the KIT abnormalities influence the white blood cell counts (P0.05) and survival (P0.04) of CML patients.

The patient was a 62-year-old man. His hematological data in April 2000 had shown no abnormalities, but he was referred to our hospital because of a fever and leukocytosis in June 2000. The peripheral blood showed 29.8 x 10(9)/L white blood cells, with 68.0% blasts. A bone marrow aspirate showed hypercellularity with a proliferation of large leukemic blasts. The leukemic cells were positive for CD13 (91%), CD33 (54.8%), CD34 (94.5%), and HLA-DR (97.9%). Some leukemic cells (15.6%) also expressed CD14. Cytogenetic analysis revealed 92,XXYY,t(9;22)(q34;q11)x2 in all 20 metaphase cells. Reverse transcriptase polymerase chain reaction analysis detected the minor BCR/ABL messenger RNA (mRNA) but failed to detect the major BCR/ABL mRNA. The patient achieved complete remission after induction chemotherapy, with no evidence of Philadelphia chromosome (Ph) or minor BCR/ABL mRNA. Ph-positive acute myeloid leukemia (Ph-AML) has rarely been reported. Herein, we report a case of Ph-AML with tetraploidy and review the previously reported Ph-AML cases.

Gene alterations accumulate during the progression of acute myelogenous leukemia (AML) to a malignant clone. Here, a new myeloid cell line, designated YSK-21, with the balanced t(8;21)(q22;q22) and the unbalanced der(1)t(1;17)(p36;q21), was established. YSK-21 grows well in a medium containing recombinant human granulocyte colony-stimulating factor (rhG-CSF), granulocyte-macrophage colony-stimulating factor (rhGM-CSF), or interleukin-3 (rhIL-3). Molecular analysis using the reverse transcriptase-polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH) revealed that t(8;21)(q22;q22) resulted in an AML1-MTG8 fusion transcript. FISH and spectral karyotyping (SKY) in conjunction with G-banding analysis revealed a der(1)t(1;17)(p36;q21) chromosomal translocation, which appeared in the clone developed from the original leukemic cells. Molecular analysis of the TP73 gene on 1p36 and the TP53 gene revealed a deletion of one-allele in TP73 with partial demethylation of another allele in the initial clone of YSK, and a point mutation consisting of an AT substitution in codon 288 of the TP53 gene in the developed clone of YSK-21. YSK-21 cells, expressing aberrant AML1-MTG8, TP53, and TP73 protein molecules, may be useful for elucidating the pathophysiology of these aberrant proteins and for studying the der(1)t(1;17)(p36;q21) chromosomal translocation. © 2001 Wiley-Liss, Inc.

The molecular mechanism of carcinogenesis is a multistep process that is characterized by both activation of oncogenes and inactivation of tumor suppressor genes. In the present study, mutations of N-ras, p53 and FMS-like tyrosine kinase 3 (FLT-3) genes and loss of expression of the deleted in colorectal carcinoma (DCC) gene were analyzed in 59 patients with myelodysplastic syndromes (MDS). Mutations of N-ras, p53, and FLT-3 genes were detected in 7, 7, 1 of the 59 patients with MDS, respectively. Loss of DCC expression was detected in 16 patients. Type of MDS patients with N-ras mutation were all refractory anemia with excess of blasts in transformation (RAEB-T). Abnormalities of p53 and DCC genes were significantly associated with survival time (p< 0.02, p< 0.004, respectively).

We report here a very rare case of chronic myeloid leukemia (CML) with a minor bcr-abl transcript, which developed following long-term chemotherapy with fluorouracil for esophageal carcinoma. A 64-year-old male patient was diagnosed with CML. Four years earlier, he had suffered from esophageal carcinoma, which was treated by surgical resection followed by oral administration of fluorouracil (200 mg/day) for 4 years. Molecular analysis of his Philadelphia chromosome (Ph) using reverse-transcriptase polymerase chain reaction (RT-PCR) and subsequent sequencing revealed a minor bcr-abl transcript. The clinical course of this patient was aggressive with a short chronic phase of CML. This is the first reported case of secondary CML with a minor bcr-abl transcript.

We report a rare large B-cell non-Hodgkin's lymphoma having a characteristic near-triploid cell population with add(17)(p22) and t(14;18)(q32;q21) translocation. We also established and characterized a new cell line (TK cell) derived from the present lymphoma. A codon 180 mutation (GAG --> GAT) in the p53 gene was detected. t(14;18)(q32;q21) was revealed juxtaposition of the bcl-2 and JH genes. Immunoprecipitation analyses of p53 and bcl-2 revealed that abnormality of the p53 protein and aberrant bcl-2 expression, which may protect cells from apoptosis, may be critical to the development of leukaemogenesis exhibiting near-triploid chromosomes.

In acute leukemia (AL) with a late-appearing Philadelphia (la-Ph) translocation, it is unclear whether these translocations arise from the same molecular event as classical Ph translocations. In order to elucidate the molecular events of la-Ph and subsequent translocations of la-Ph leukemia, we performed molecular analysis on the complex rearrangements, in a cell line, MY, which was established from bone marrow mononuclear cells of a patient with a la-Ph acute biphenotypic leukemia. This la-Ph, expressing an acute lymphoblastic leukemia (ALL)-type BCR/ABL transcript, produces a novel P180BCR/ABL fusion protein reflecting deletion of 174 bases (58 amino acids) encoded by the a2 exon of the ABL gene. An immune complex kinase assay showed that this protein had autophosphorylation activity. Fluorescence in situ hybridization (FISH) in conjunction with G-banding analysis revealed that the initial der(9)t(9;22)(q34;q11) progressed to a der(9)(9pter-->9q34::22q11-->22q13::5q11.2 -->5q15:: 10q23-->10qter) by, first, a three-way translocation among the der(9)t(9;22)(q34;q11), chromosome 5, and the normal chromosome 22, and then a subsequent translocation with chromosome 10. Moreover, both the end-stage leukemic cells of the patient and the MY cell line had another translocation, t(X;12)(p11.2;p13). The 12p breakpoint was located near the ETV6 gene by analysis of pulsed-field gel electrophoresis, but transcription of ETV6 was unaffected. Tumorigenicity analysis indicated that an additional translocation, t(2;3)(p16;q29), may have caused a more malignant clone, because only MY cells with the t(2;3)(p16;q29) were capable of growing subcutaneously in nude mice within 40 days. The molecular events of leukemogenesis and leukemic progression in the present la-Ph AL occurred by accumulation of unique translocations. This cell line, MY, expressing a novel variant P180BCR/ABL protein with a deletion of the a2 exon of the ABL gene, may be useful for elucidating the pathophysiology of this fusion protein and for studying ETV6-related leukemogenesis and t(2;3), as well as the molecular mechanisms of the complex translocations.

There are some types of leukemia and lymphoma, especially T-cell types, that show skin infiltration and possibly cause erythroderma. Recently, the International Society for Cutaneous Lymphomas (ISC...

A number of CML patients who achieve a sustained complete molecular response (CMR) for at least 2 years during imatinib (IM) therapy can discontinue IM without relapse. With the long-term goal of developing immunological criteria for managing IM therapy in CML patients, we compared the immunophenotypic profiles of three groups of CML patients: those who received IM and had a CMR for more than two consecutive years (CMR group); patients who received IM and did not have a sustained CMR but maintained a major molecular response for more than 2 years (fluctuating CMR group); and patients with a sustained CMR for more than 6 months after IM discontinuation (STOP-IM group), together with healthy controls. The percentages of effector populations of natural killer (NK) cells, such as interferon (IFN)-γ(+) CD3(-) CD56(+) cells, were significantly higher in the STOP-IM and CMR groups than in the fluctuating CMR and control groups. The elevated levels of these effector NK cells were sustained for more than 3 years after IM discontinuation. In contrast, the percentages of effector memory CD8(+) T cells, such as IFN-γ(+) CCR7(-) CD45RO(+) CD8(+) cells, were significantly higher in the STOP-IM and control groups than in the CMR and fluctuating CMR groups, possibly owing to IM intake. These results suggest that the immunological activation status of NK cells contributes to CMR maintenance. Higher activation levels of effector NK cells in CML patients being treated with IM might reflect minimization of BCR-ABL1 transcript levels and therefore could be additive information for determining whether to stop IM.

Inactivation of the deleted in colorectal carcinoma (DCC) tumor suppressor gene has been reported not only in colorectal carcinoma but also in other human malignancies. In order to evaluate the role of the DCC gene in leukemogenesis, we examined DCC expression using the reverse transcriptase-polymerase chain reaction (RT-PCR) method. Expression of the DCC gene was reduced or absent in 10 of 39 (26%) patients with acute myelogenous leukemia (AML), three of 14 (29%) patients with acute lymphocytic leukemia (ALL), seven of 33 (21%) patients with chronic myelogenous leukemia (CML), three of 39 (8%) patients with myelodysplastic syndromes (MDS), and five of nine (56%) patients with overt leukemia progressed from MDS. These findings suggest that inactivation of the DCC gene contributes to some instances of leukemogenesis.

Mixed-lineage leukemia (MLL)/AF4-positive acute lymphoblastic leukemia (ALL) is a common type of leukemia in infants, which is associated with a high relapse rate and poor prognosis. IL24 selectively induces apoptosis in cancer cells and exerts immunomodulatory and antiangiogenic effects. We examined the effects of adeno-associated virus type 8 (AAV8) vector-mediated muscle-directed systemic gene therapy in MLL/AF4-positive ALL using IL24. In a series of in vitro studies, we examined the effects of AAV8-IL24–transduced C2C12 cell-conditioned medium. We also examined the effects of AAV8-IL24 in MLL/AF4 transgenic mice. The results revealed the effects of AAV8-IL24 in MLL/AF4-positive ALL both in vitro and in vivo. With regard to the mechanism of therapy using AAV8-IL24 in MLL/AF4-positive ALL, we demonstrated the antiangiogenicity and effects on the ER stress pathway and unreported pathways through inhibition of S100A6 and HOXA9, which is specific to MLL/AF4-positive ALL. Inhibition of S100A6 by IL24 was dependent on TNF-α and induced acetylation of p53 followed by activation of the caspase 8–caspase 3 apoptotic pathway. Inhibition of HOXA9 by IL24, which was independent of TNF-α, induced MEIS1 activation followed by activation of the caspase 8–caspase 3 apoptotic pathway. Thus, gene therapy using AAV8-IL24 is a promising treatment for MLL/AF4-positive ALL.